Epo is a cytokine that stimulates erythropoiesis and is also known as a therapeutic agent for renal anemia, having achieved satisfactory treatment for a long time. Epo is mainly produced in the liver (embryonic stage) and the kidney (after birth) of mammals. In chronic renal dysfunction, the Epo production in the kidney decreases to cause anemia (renal anemia) (Non Patent Literature 1). This anemia is cured by administration of Epo prepared by a genetic recombination technology, and thereby the prognosis and the quality of life of renal anemia patients have been dramatically improved.
It has been revealed that Epo in tissues, such as the central nervous system, the retina, the kidney, and the heart, shows cytoprotection against stress, and the clinical use of Epo intended to have an effect other than hematopoiesis has been started in some clinical fields. At the same time, it is also reported, for example, that administration of Epo also stimulates proliferation of cancer cells to cause a decrease in performance of treatment and that administration of Epo facilitates thrombus formation to shorten the lifetime. In the new guidelines of the USA, more strict standards apply to administration of Epo and similar drugs. However, data, such as those of animal experiments, supporting these effects of Epo are insufficient, except for clinical statistical data, and establishment of a useful experimental system has been therefore demanded.
Incidentally, a hypoxia-inducible factor (HIF) has been identified by research on controlling expression of an Epo gene using a hepatocyte-derived cell line, and this factor is now recognized as an important factor in various biological activities such as inflammation, genesis, and carcinogenesis as well as energy metabolism. In the kidney, only a small number of specific cells produce Epo, and it is predicted that an unknown factor regulating tissue specificity and hypoxia reactivity works in the production, and elucidation thereof has high possibility of leading to elucidation of pathological conditions of various diseases and development of a method of treatment. However, the production of Epo is suppressed to be very low under a usual breeding environment. Therefore, Epo-producing cells cannot be identified or analyzed without anemia induction by blood removal or phenylhydrazine administration or stimulation of Epo production, such as breeding in a hypoxia chamber, and such a situation has continued for a long period of time.
Furthermore, there are many reports on control of proliferation and survival of cells by Epo, but research on the differentiation induction mechanism by Epo is few. In the time when Epo was found, it was believed that a receptor of Epo is specifically present in erythroid cells and that Epo mainly functions for supporting the survival of the cells. In the subsequent research, it was believed that Epo may also actively affect the program of erythroid differentiation. However, the detailed mechanism has not been elucidated. Incidentally, erythropoiesis associated with an increase in blood Epo concentration, such as under a high altitude or anemia, is called stress hematopoiesis, and, in mice, the spleen is the place for the hematopoiesis. In general, the Epo concentration in regular hematopoiesis is kept very low. Accordingly, it is predicted that the molecular mechanism of the stress hematopoiesis is different from that of the regular hematopoiesis, but the detail thereof is unclear. Furthermore, in chronic anemia associated with inflammation or cancer, the Epo production and Epo sensitivity decrease, but the mechanism thereof is also unclear.
In order to solve these problems, development of mice with adult-onset deficiency in Epo production has been demanded. Conventionally, in research on renal anemia and acquisition of experimental animals less producing Epo, methods in which the function of the kidney is reduced to reduce the Epo production have been used (Non Patent Literatures 2 to 4). In this method, the majority of the kidney of a rat is removed to induce a decrease in Epo production and anemia associated with deterioration of renal function, which requires skill in, for example, the operative procedure and is a procedure taking a long time. Thus, acquisition of sufficient populations of such animals required much labor.
Epo gene knockout mice develop severe anemia and die in the embryonic stage and, therefore, could not be used in analysis of such a purpose (Non Patent Literature 5). EpoGFP/+ mice were created by knockin of a green fluorescent protein (GFP) gene into the Epo gene to label the Epo-producing cells, and heterozygous mice were crossed to each other to investigate the phenotype of EpoGFP/GFP mice. The phenotype of the EpoGFP/GFP mice was almost the same as that of the Epo gene knockout mice first reported and the EpoGFP/GFP mice died by the 13th day of embryonic life (Non Patent Literature 6).
In order to investigate the functions of Epo in adult mice, conditional knockout mice were produced (Non Patent Literature 6). The mice were produced by rescuing EpoGFP/GFP mice from lethality with an Escherichia coli artificial chromosome (bacterial artificial chromosome: BAC) containing an Epo gene carrying a loxP sequence, and embryonic lethality was completely reproduced by crossing with mice broadly expressing Cre recombinase. In addition, expression of drug-induced CRE in the rescued mice caused a decrease in Epo expression in the adult mice. Another laboratory also reported conditional knockout mice produced by directly inserting a loxP sequence into an Epo gene to induce the action of a drug-induced Cre recombinase (Non Patent Literature 7). However, in both experiments, since recombination of the Epo gene in every Epo-producing cell in the body is difficult, the anemia of the mice bred under a usual environment was mild, Epo production induced by anemia induction was recognized, and an increase in reactive hematocrit similar to that of a wild-type was recognized. Furthermore, drug administration has been started after weaning, and analysis can be performed at the point of time when the influence of the drug has completely disappeared after completion of the drug administration. Therefore, in experiments requiring a large number of populations, a large amount of labor and time are necessary. Regarding a HIF2 gene, which is indispensable for hypoxia inducibility control of Epo production, conditional knockout mice were reported (Non Patent Literature 8), and the phenotype thereof was almost the same as that of the Epo gene conditional knockout mice.
In 1993, Maxwell, et al. reported a transgenic mouse produced by inserting the T antigen gene of SV40 virus into an Epo gene fragment of 16 kb, in which homologous recombination of a transgene unexpectedly occurred in the 5′ untranslated region of an erythropoietin gene locus (EPO-TAgh) (Non Patent Literature 9). In this mouse, the T antigen mRNA of SV40 is transcribed following a part of the 5′ UTR sequence of Epo mRNA. Though the detail of gene construction on the 3′ side is unclear, since Epo production detectable by ELISA is present, it is predicted that small amounts of mRNA and protein of the site where the Epo gene is translated are produced. The plasma Epo concentration of the mouse has been reported to be 55±18 pg/mL (wild type: 122±16 pg/mL) under a usual environment and is thus lower than that of the wild type. However, in breeding in a hypoxia chamber, the Epo concentration was 162±25 pg/mL (wild type: 460±39). The concentration significantly increased, although the increase was lower than that of the wild type, and it was judged that the hypoxia inducibility of the Epo gene expression was maintained (Non Patent Literature 10). In the first report, the homozygous mice had a hematocrit of 13.2±3.3% and had severe anemia (Non Patent Literature 9). In the subsequent report on Epo TAg, the hematocrit of homozygous mice was 19.2±0.2% (Non Patent Literature 11). Also in other reports, the values were similar levels (Non Patent Literatures 10, 12, and 13). The hematocrit of EPO-TAgh heterozygote was reported to be 34.4±3.5%, which suggests a possibility that in EpoTAg heterozygote, a material produced from the EPO-TAgh gene locus dominant-negatively acts on the wild-type Epo to cause anemia. EPO-TAgh homozygous mice are used in research on, for example, genetic treatment. In addition, immunological response to mouse Epo is also reported, and it is predicted that the immune system is activated for expressing SV40 TAg having strong immunogenicity (Non Patent Literature 11). It is concerned from these results that too many unclear points remain in the Epo-TAg mouse to use it as a standard of Epo production deficient mouse.
It was reported that Epo-producing cells (renal Epo producing cells: REPs) of the kidney, which is a main organ producing Epo in adults, fibroblast-like cells present so as to surround the proximal tubule and express a neuronal marker, by producing a transgenic mouse, Epo-BAC-GFP-Tg, having a bacterial artificial chromosome (BAC) containing an Epo gene and identifying the Epo-producing cells using both the Epo-BAC-GFP-Tg and Epo (GFP/+) mice (Non Patent Literatures 14 and 15).
Many of past papers regarding the mechanism of controlling Epo production are based on research using a liver cancer-derived cell line, and the characteristics of REPs and the surrounding environment thereof are completely different from those of hepatocytes. In the kidney, there is a possibility that an Epo production-controlling mechanism different from that in the liver is working. The Epo-BAC-GFP-Tg mouse is an excellent experimental animal for analyzing Epo production control in the kidney, but requires treatment, such as induction of anemia by successive blood-removing treatment or breeding in a hypoxia chamber, in actual research. In addition, the anemia induced by such treatment has the disadvantage of a large individual difference.